Populations of surface-attached microorganisms are commonly referred to as biofilms. In most natural settings bacteria are found predominantly in biofilms, yet for many years studies of bacterial physiology focused on the planktonic state of bacterial cells. The widespread recognition that biofilms impact a myriad of environments has led to concerted efforts to gain a better understanding of the molecular mechanisms that underlie the development of these communities. Recent results have revealed that biofilm formation is a complex developmental process that occurs in response to environmental cues. Working models for how planktonic bacteria proceed from environmental sensing to the formation of mature biofilms are now guiding many investigators in their research. However, most of the attention has been placed on biofilms that form on abiotic solid surfaces such as plastics and glass. The formation of biofilms on living surfaces is also widespread and has important impacts in environmental and clinical settings. In our first experimental approach we propose to extend the studies we have carried out with a model bacterium, Pseudomonas aeruginosa, to investigate how it forms a biofilm on living fungal filaments. To this end we will: 1) Characterize the bacterial-fungal interactions through physiological, biochemical and microscopic analyses, 2) Select specific genes in which to generate mutations and test their effects on bacterial-fungal interactions and 3) Carry out a mutant screen and perform transcriptional profiling using microarrays to identify additional genes involved in the bacterial-fungal interactions. It is generally accepted that there is cellular differentiation within biofilms. Yet, relatively little is known about the molecular mechanisms that underlie differentiation processes in bioflims. In a second experimental approach that follows a path analogous to our first approach, we will address the question of cellular differentiation in biofilms through the study of a well-characterized sporulation process in Bacillus subtilis. We will: 1) Analyze the spatial and temporal patterns of transcription of a spore-specific gene using reporter fusions and light, electron and confocal scanning laser microscopic techniques, 2) Generate additional reporter gene fusions to selected genes and analyze their spatial and temporal patterns of expression in order to develop a more complete functional anatomy of the biofilm and 3) Test the effects of mutations involved in cell-cell signaling and environmental sensing on cellular differentiation within the biofilm.